Suppression of immune response via inhibition of cathepsin S

ABSTRACT

Methods and products for suppressing a class II MHC-restricted immune response in a mammal, or in mammalian cells, are described. The methods depend upon inhibiting invariant chain proteolysis by cathepsin S from class II MHC/invariant chain complexes, thereby reducing the competency of Class II MHC molecules for binding antigenic peptides, reducing presentation of antigenic peptides by class II MHC molecules, and suppressing immune responses. The methods may be employed in the treatment of autoimmune diseases, allergic responses, and organ or tissue graft rejection. Pharmaceutical and therapeutic compositions which are peptide-based inhibitors of cathepsin S are also described.

RELATED APPLICATIONS

This application claims benefit of priority to U.S. ProvisionalApplication No. 60/018,100 filed Apr. 22, 1996.

GOVERNMENT SUPPORT

The present invention was supported, in part, by grant 5-R01-A134893from the National Institutes of Health and grant 5-R01-AI38577 from theNational Institutes of Health. Therefore, the government may havecertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel methods and products directed toimmunosuppression via the inhibition of cathepsin S. The methods andproducts may by employed for the treatment of autoimmune diseases, aswell as reducing the competency of class II MHC molecules for bindingantigenic peptides.

BACKGROUND OF THE INVENTION

Class II MHC (major histocompatibility complex) cellular proteins (αβheterodimers) associate early during biosynthesis with a type IImembrane polypeptide, the invariant chain (Ii), to form class IIMHC/invariant chain complexes (αβIi). It has been reported that theinvariant chain associates with class II MHC molecules via directinteraction of residues 81-104 of its lumenal domain, designated classII associated invariant chain peptides (CLIP), with the antigen bindinggroove of class II MHC.

The invariant chain contains a signal in its cytoplasmic tail whichdelivers the class II MHC/invariant chain complexes to intracellularendocytic compartments, where the class II MHC molecules encounter andbind antigenic peptides. A prerequisite for antigenic peptide loading ofclass II MHC molecules is the proteolytic destruction of the invariantchain from the class II MHC/invariant chain complexes. Identification ofthe specific key protease responsible for this proteolysis has notpreviously been reported. Proteolysis of the invariant chain allows theantigenic peptides to bind to the class II MHC molecules to form classII MHC/antigenic peptide complexes.

The antigenic peptides in these complexes are then deposited on the cellsurface for recognition by CD4+T cells. These T cells are involved inthe production of cytokines and thus help orchestrate an immuneresponse, culminating in the appropriate production of antibodies. Onoccasion, CD4+cells are activated inappropriately and are believed tocontribute to the pathology of autoimmune disease.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for inhibitinginvariant chain proteolysis from class II MHC/invariant chain complexes,reducing the competency of class II MHC molecules for binding antigenicpeptides, and reducing the presentation of antigenic peptide by class IIMHC molecules, by administering to a mammalian cell, in vivo or invitro, an amount of an inhibitor of cathepsin S effective tosubstantially inhibit proteolysis of invariant chain by cathepsin S.

In another aspect, the present invention provides methods for modulatingclass II MHC-restricted immune responses. Such immune responses areessential to autoimmune diseases, allergic reactions, and allogeneictissue rejections. Therefore, the present invention also providesmethods for suppressing class II MHC-restricted immune responses and, inparticular, autoimmune, allergic, and allogeneic immune responses, byadministering to a mammal (e.g., a human patient) a therapeuticallyeffective amount of an inhibitor of cathepsin S to reduce thepresentation of antigenic peptides by class II MHC molecules and,thereby, provide a degree of relief from these conditions. In preferredembodiments, methods are provided for the treatment of autoimmunediseases including juvenile onset diabetes (insulin dependent), multiplesclerosis, pemphigus vulgaris, Graves' disease, myasthenia gravis,systemic lupus erythematosus, rheumatoid arthritis and Hashimoto'sthyroiditis. In other preferred embodiments, methods are provided fortreating allergic responses, including asthma, and for treatingallogeneic immune response, including those which result from organtransplants, including kidney, lung, liver, and heart transplants, orfrom skin or other tissue grafts.

The inhibitors of cathepsin S may be any molecular species which inhibitthe transcription of a cathepsin S gene, the processing or translationof a cathepsin S mRNA, or the processing, trafficking or activity of acathepsin S protein, when administered in vivo or in vitro to amammalian cell which is otherwise competent to express active cathepsinS. In particular, inhibitors may be repressors, or antisense sequences,or competitive and non-competitive inhibitors such as small moleculeswhich structurally mimic the natural substrates of cathepsin S but whichare resistant to the proteolytic activity of the enzyme, or antibodies,ribozymes, and the like. Preferably, the inhibitors are cysteineprotease inhibitors.

In preferred embodiments, the cathepsin S inhibitors are “selective”inhibitors of cathepsin S which fail to inhibit, or inhibit to asubstantially lower degree, at least one of cathepsins K, L, H, O2 andB, and in most preferred embodiments, the inhibitors are “specific”inhibitors of cathepsin S which fail to inhibit, or inhibit to asubstantially lower degree, each of cathepsins K, L, H, O2 and B.

In addition, preferred inhibitors include peptide-based inhibitors whichmimic a portion of a naturally occurring cathepsin S substrate. Suchpeptide based inhibitors include peptidyl aldehydes, nitriles,α-ketocarbonyls, halomethyl ketones, diazomethyl ketones,(acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts,epoxides, and N-peptidyl-O-acyl-hydroxylamines. Preferred peptide-basedinhibitors of cathepsin S also include those based upon the sequencesLeu-Leu-Leu, and Leu-Hph, such as Leu-Leu-Leu-vinyl sulfone,N-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone,N-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone, andmorpholinurea-Leu-Hph-vinylsulfone phenyl (LHVS).

In another aspect, the present invention provides a new class ofpeptide-based inhibitors of cathepsin S based upon the newly disclosedpreferred chain cleavage site spanning from N-terminally about positions68-75 to C-terminally about positions 83-90 of the invariant chainsequence. Thus, peptide-based inhibitors of cathepsin S based upon asequence of 2-20, more preferably 2-10, and most preferably 2-3consecutive residues from within this site are provided. Particularlypreferred are peptide-based inhibitors of cathepsin S based upon thesequences Asn-Leu, Glu-Asn-Leu, Arg-Met, and Leu-Arg-Met (positions 77,78, and 79, or −3, −2 and −1 relative to the Lys₈₀ cleavage point) arepreferably used as a basis for choosing or designing a peptide-basedinhibitor.

Thus, for example, the invention provides novel peptide-based inhibitorssuch as vinylsulfone compounds including Asn-Leu-vinylsulfone,Arg-Met-vinylsulfone, Leu-Arg-Met-vinylsulfone, andGlu-Asn-Leu-vinylsulfone. Modifications of these peptide vinylsulfonesare also included in the invention. For example, carboxybenzyl can bepresent at the N-terminal end to give the following compounds:N-(carboxybenzyl)-Asn-Leu-vinylsulfone,N-(carboxybenzyl)-Arg-Met-vinylsulfone,N-(carboxybenzyl)-Leu-Arg-Met-vinylsulfone, andN-(carboxybenzyl)-Glu-Asn-Leu-vinylsulfone. In an alternative,nitrophenylacetyl is present at the N-terminal end to give the followingcompounds: N-(nitrophenylacetyl)-Asn-Leu-vinylsulfone,N-(nitrophenylacetyl)-Arg-Met-vinylsulfone,N-(nitrophenylacetyl)-Leu-Arg-Met-vinylsulfone, andN-(nitrophenylacetyl)-Glu-Asn-Leu-vinylsulfone.

The invention is also meant to include other peptide-based inhibitorsbased on the peptide sequences of the preferred invariant chain cleavagesite of cathepsin S, including peptidyl aldehydes, nitriles,α-ketocarbonyls, halomethyl ketones, diazomethyl ketones,(acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts,epoxides, and N-peptidyl-O-acyl-hydroxylamines, and those with variousother substitutions at the amino terminus as would be known to thoseskilled in the art.

Other embodiments of the present invention will be apparent to one ofordinary skill is in the art from the foregoing and from the DetailedDescription and Examples presented below.

DETAILED DESCRIPTION

The present invention is based, in part, upon the discovery that themammalian cysteine protease cathepsin S is of primary importance in theproteolysis of invariant chain polypeptides complexed to class II ARC αβheterodimers. In particular, it is herein disclosed that cathepsin Sappears to be responsible for the normal cleavage of the invariant chainpolypeptide while it is associated in class II MHC/invariant chaincomplexes and, therefore, that inhibition of cathepsin S inhibits theproteolysis of invariant chain from class II MHC/invariant chaincomplexes are inhibits the formation of class II MHC/CLIP complexes.Consequently, because class II MHC molecules remain associated in classII MHC/invariant chain complexes, inhibition of cathepsin S reduces thecompetency of class II MHC molecules for binding antigenic peptides andreduces the presentation of antigenic peptides by class II MHCmolecules.

Therefore, in one aspect, the present invention provides for methods forinhibiting;g; invariant chain proteolysis from class II MHC/invariantchain complexes, reducing the competency of class II MHC molecules forbinding antigenic peptides, and reducing the presentation of antigenicpeptide by class II MHC molecules, by administering to a mammalian cell,in vivo or in vitro, an amount of an inhibitor of cathepsin S effectiveto substantially inhibit proteolysis of invariant chain by cathepsin S.

In another aspect, because presentation of antigenic peptides complexedwith class II MHC molecules is essential to immune responses which areclass II MHC-restricted, the present invention also provides methods formodulating class II MHC-restricted immune responses. Such immuneresponses are essential to autoimmune diseases, allergic reactions, andallogeneic tissue rejections. Therefore, the present invention alsoprovides methods for suppressing class II MHC-restricted immuneresponses and, in particular, autoimmune, allergic, and allogeneicimmune responses, by administering to a mammal (e.g., a human patient) atherapeutically effective amount of an inhibitor of cathepsin S toreduce the presentation of antigenic peptides by class II MHC moleculesand, thereby, provide a degree of relief from these conditions.

The present invention is further based, in part, upon the discovery thatcathepsin S normally acts on class II MHC/invariant chain complexes atone of two, nearly adjacent, preferred invariant chain cleavage sites.In particular, it is herein disclosed that human cathepsin S normallycleaves the invariant chain at the peptide bonds C-terminal to residuesArg₇₈ and Lys₈₀ of the invariant chain sequence (SEQ ID NO: 1).

Therefore, in another aspect, the present invention provides for a newclass of peptide-based inhibitors of cathepsin S. These peptide-basedcathepsin S inhibitors are based upon the amino acid residue sequencesimmediately surrounding the cleavage sites which are recognized, bound,and cleaved by cathepsin S (e.g., residues 68-90, or 73-85 of SEQ ID NO:1). The peptide-based cathepsin S inhibitors may be actual peptides or,more preferably, peptide derivatives or modified peptides which retainsufficient structural similarity to the natural substrate to retainbinding activity, but which may be structurally modified to render themnon-competitive inhibitors or to otherwise enhance their stability.

Preferred embodiments and exemplifications of the present invention aredescribed in detail below.

I. Definitions

In order to more clearly and concisely describe and disclose the subjectmatter of the claimed invention, the following definitions are providedfor specific terms used in the specification and appended claims.

As used herein, an “inhibitor of cathepsin S” is any molecular specieswhich inhibit, the transcription of a cathepsin S gene, the processingor translation of a cathepsin S mRNA, or the processing, trafficking oractivity of a cathepsin S protein, when administered in vivo or in vitroto a mammalian cell which is otherwise competent to express activecathepsin S. Thus, for example, the term “inhibitor of cathepsin S”embraces a repressor which inhibits induction and/or transcription ofthe cathepsin S gene, or an antisense sequence which selectively bindsto cathepsin S DNA or mRNA sequences and which inhibits thetranscription or translation (if the cathepsin S sequences. Similarly,the term “inhibitor of cathepsin S” includes competitive andnon-competitive inhibitors of the activity of the cathepsin S protein,such as small molecules which structurally mimic the natural substratesof cathepsin S but which are resistant to the proteolytic activity ofthe enzyme. Although an inhibitor of cathepsin S may have some degree ofinhibitory activity for other genes or proteins which are structurallyor functionally related, the term “inhibitor of cathepsin S” is notintended to embrace non-selective suppressors of all gene expression orprotein synthesis, or general toxins (e.g., transcription blockers suchas actinomycin D, and α-amanitin, protein synthesis inhibitors such aspuromycin, cycloheximide, and diptheria toxin).

As used herein, a “cysteine protease inhibitor” is any molecular specieswhich inhibits one or more of the mammalian enzymes known as cysteineproteases and, in particular, which inhibits cathepsin S. The cysteineproteases, which are also known as thiol or sulfhydryl proteases orproteinases, are proteolytic enzymes with active site cysteine residueswhich act as nucleophiles during catalysis. Cysteine proteases includepapain, calpain I, calpain II, cruzain, and cathepsins S, K, L, H, O2and B. (Note that cathepsin D is an aspartyl protease.)

As used herein, a “selective inhibitor of cathepsin S” is any molecularspecies which, as defined above, is an inhibitor of cathepsin S butwhich fails to inhibit, or inhibits to a substantially lower degree, atleast one of cathepsins K, L, H, O2 and B. In preferred embodiments, aselective inhibitor of cathepsin S is employed which has a second orderrate constant of inactivation or inhibition for cathepsin S which is atleast twice and, more preferably, five times higher than thecorresponding rate constant for at least one of cathepsins K, L, H, O2and B. Most preferably, a selective inhibitor of cathepsin S has asecond order rate constant of inactivation for cathepsin S which is atleast an order of magnitude or, most preferably, at least two orders ofmagnitude higher than its inactivation rate constant for at least one ofcathepsins K, L, H, O2 and B. As used herein, the term “second orderrate constant of inactivation” is intended to mean that quantity asknown in the art, and represented as k_(inact)/K_(i) or as k₂/K_(i).See, e.g., Brömme et al., Biol. Chem. 375:343-347 (1994); Palmer et al.,J. Med. Chem. 38:3193-3196 (1995); Brömme et al., Biochem. J. 315:85-89(1996).

As used herein, a “specific inhibitor of cathepsin S” is any molecularspecies which, as defined above is an inhibitor of cathepsin S but whichfails to inhibit, or inhibits to a substantially lower degree each ofcathepsins K, L, H, O2 and B. In preferred embodiments, a specificinhibitor of cathepsin S is employed which has a second order rateconstant of inactivation for cathepsin S which is at least twice and,more preferably, five times higher than the corresponding rate constantsfor each of cathepsins K, L, H, O2 and B. Most preferably, a specificinhibitor of cathepsin S has a second order rate constant ofinactivation for cathepsin S which is at least an order of magnitude or,most preferably, at least two orders of magnitude higher than its secondorder rate constants for each of cathepsins K, L, H, O2 and B.

As used herein with respect to class II MHC-restricted immune responses,“suppressing” means reducing in degree or severity, or extent orduration, the overt manifestations of the immune response including, forexample, reduced binding and presentation of antigenic peptides by classII MHC molecules, reduced activation of T-cells and B-cells, reducedhumoral and cell-mediated responses and, as appropriate to theparticular immune response, reduced inflammation, congestion, pain, ornecrosis. “Suppression” of an immune response does not require completenegation or prevention of any of these manifestations of an immuneresponse, but merely a reduction in degree or severity, or extent orduration, which is of clinical or other practical significance.

As used herein with respect to inhibitors of cathepsin S, the terms“peptide-based” and “non-peptide-based” do not mean that an inhibitordoes, or does not, comprise a peptide or polypeptide, but that thestructure of the inhibitor is based upon, or is not based upon, thestructure of a polypeptide sequence which binds as a substrate in theactive site of cathepsin S.

II. Inhibition of Invariant Chain Proteolysis. Antigen Binding, andAntigen Presentation

As noted above, and as evidenced in the examples below, cathepsin S isbelieved to be important to normal proteolytic processing of theinvariant chain. Therefore, in one aspect, the present inventionprovides methods for inhibiting invariant chain proteolysis from classII MHC/invariant chain complexes in mammalian cells, in vivo or invitro, by administering an of cathepsin S to the cells. As a result ofthe inhibition of cathepsin S, proteolysis of the invariant chain fromclass II MHC/invariant chain complexes within the cells is alsoinhibited.

Furthermore, under normal physiological conditions, inhibition of theproteolysis of the invariant chain inhibits formation of class IIMHC/CLIP (also referred to as αβ-CLIP) complexes. Therefore, as class IIMHC/CLIP complexes are more readily loaded with antigenic peptides thanclass II MHC/invariant chain complexes, the inhibition of cathepsin Sand consequent inhibition of MHC/CLIP complex formation reduces thecompetency of class II MHC molecules for binding antigenic peptides.Therefore, in one aspect, the present invention provides methods forreducing the competency of class II MHC molecules for binding antigenicpeptides in mammalian cells, in vivo or in vitro, by administering aninhibitor of cathepsin S to the cells. As a result of inhibition ofcathepsin S, proteolysis of the invariant chain is inhibited, formationof class II MHC/CLIP complexes is inhibited, and the competency of theMHC molecules to bind antigenic peptides is reduced.

Similarly, under normal physiological conditions, inhibition of theproteolysis of the invariant chain inhibits the loading and presentationof class II MHC molecules with antigenic peptides. Thus, as class IIMHC/CLIP complexes are more readily loaded with antigenic peptides thanclass II MHC/invariant chain complexes, the inhibition of cathepsin Sand consequent inhibition of MHC/CLIP complex formation reduces theloading and presentation of antigenic peptide by class II MHC molecules.Therefore, in one aspect, the present invention provides methods forreducing the presentation of antigenic peptides by class II MHCmolecules in mammalian cells, in vivo or in vitro, by administering aninhibitor of cathepsin S to the cells. As a result of inhibition ofcathepsin S, proteolysis of the invariant chain is inhibited, formationof class II MHC/CLIP complexes is inhibited, and the loading andpresentation of antigenic peptides by class II MHC molecules is reduced.

When employed with mammalian cells in vitro, such methods have utilityin preventing the loading of MHC molecules with antigenic peptides andin the production of empty MHC molecules. Empty MHC molecules haveutility for subsequent loading and use as analytical, diagnostic andtherapeutic agents. Alternatively, by exposing such cells in culture tohigh concentrations of desired antigenic peptides, or precursors of suchpeptides, a large proportion of MHC molecules loaded with the desiredpeptides may be produced. Class II MHC molecules selectively loaded withparticular peptides also have utilities in analytical, diagnostic andtherapeutic applications.

In these methods, an inhibitor of cathepsin S is administered to,provided to, or contacted with the cells in any manner which allows theinhibitor to enter the cells and inhibit cathepsin S. When employed invitro, the inhibitors are typically added to the cell culture medium,although microinjection may be employed if desired. When employed invivo, the inhibitors may be administered as described below in relationto the therapeutic methods.

III. Suppression of Class II MHC-Restricted Immune Responses

As noted above, and as evidenced in the examples below, cathepsin S isbelieved to be important to important to normal class II MHC-restrictedimmune responses in mammals. Therefore, in one aspect, the presentinvention provides methods for suppressing class II MHC-restrictedimmune responses in mammals by administering an inhibitor of cathepsin Sto the mammal. As a result of cathepsin S inhibition, the proteolysis ofinvariant chains, formation of class II MHC/CLIP complexes, and loadingand presentation of antigenic peptides are inhibited and, therefore,class II MHC-restricted immune response is suppressed.

In one series of embodiments, the methods are employed to treat mammals,particularly humans, at risk of, or afflicted with, autoimmune disease.By autoimmunity is meant the phenomenon in which the host's immuneresponse is turned against its own constituent parts, resulting inpathology. Many human autoimmune diseases are associated with certainclass II MHC-complexes. This association occurs because the structuresrecognized by T cells, the cells that cause autoimmunity, are complexescomprised of class II MHC molecules and antigenic peptides. Autoimmunedisease can result when T cells react with the host's class II MHCmolecules when complexed with peptides derived from the host's own geneproducts. If these class II MHC/antigenic peptide complexes areinhibited from being formed, the autoimmune response is reduced orsuppressed. Any autoimmune disease in which class II MHC/antigenicpeptide complexes play a role may be treated according to the methods ofthe present invention. Such autoimmune diseases include, e.g., juvenileonset diabetes (insulin dependent), multiple sclerosis, pemphigusvulgaris, Graves' disease, myasthenia gravis, systemic lupuserythematosus, rheumatoid arthritis and Hashimoto's thyroiditis.

In another series of embodiments, the methods are employed to treatmammals, particularly humans, at risk of, or afflicted with, allergicresponses. By “allergic response” is meant the phenomenon in which thehost's immune response to a particular antigen is unnecessary ordisproportionate, resulting in pathology. Allergies are well known inthe art, and the term “allergic response” is used herein in accordancewith standard usage in the medical field. Examples of allergies include,but are not limited to, allergies to pollen, “ragweed,” shellfish,domestic animals (e.g., cats and dogs), bee venom, and the like. Anotherparticularly contemplated allergic response is that which causes asthma.Allergic responses may occur, in part, because T cells recognizeparticular class II MHC/antigenic peptide complexes. If these class IIMHC/antigenic peptide complexes are inhibited from being formed, theallergic response is reduced or suppressed. Any allergic response inwhich class II MHC/antigenic peptide complexes play a role may betreated according to the methods of the present invention. Although itis not expected that immunosuppression by the methods of the presentinvention will be a routine prophylactic or therapeutic treatment forcommon allergies, severe or life-threatening allergic responses, as mayarise during asthmatic attacks or anaphylactic shock, may be treatedaccording to these methods.

In another series of embodiments, the methods are employed to treatmammals, particularly humans, which have undergone, or are about toundergo, an organ transplant or tissue graft. In tissue transplantation(e.g., kidney, lung, liver, heart) or skin grafting, when there is amismatch between the class II MHC genotypes (HLA types) of the donor andrecipient, there may be a severe “allogeneic” immune response againstthe donor tissues which results from the presence of non-self orallogeneic class II MHC molecules presenting antigenic peptides on thesurface of donor cells. To the extent that this response is dependentupon the formation of class II MHC/antigenic peptide complexes,inhibition of cathepsin S may suppress this response and mitigate thetissue rejection. An inhibitor of cathepsin S can be used alone or inconjunction with other therapeutic agents, e.g., as an adjunct tocyclosporin A and/or antilymphocyte gamma globulin, to achieveimmunosuppression and promote graft survival. Preferably, administrationis accomplished by systemic application to the host before and/or aftersurgery. Alternatively or in addition, perfusion of the donor organ ortissue, either prior or subsequent to transplantation or grafting, maybe effective.

In order to minimize the potential for undesired side effects, it ispreferred in each of the above-described embodiments that an inhibitorof cathepsin S is chosen which is a selective inhibitor of cathepsin S,a specific inhibitors of cathepsin S, or a highly specific inhibitor ofcathepsin S. Thus, for example, it may not be desirable to inhibit, evenbriefly, all proteases or all cysteine proteases in a cell or anorganism because normal protein processing and turnover will bedisrupted. To the extent that such incidental inhibition is deleteriousor undesired, the use of increasingly more selective cathepsin Sinhibitors may be preferred. The use of more selective cathepsin Sinhibitors may, for example, allow for the use of higher dosages or moreextended treatment periods.

Administration of the inhibitor can be accomplished by any method whichallows the inhibitor to reach the target cells, e.g., class II MHCantigen presenting cells. These methods include, e.g., injection,infusion, deposition, implantation, anal or vaginal supposition, oralingestion, inhalation, topical administration, or any other method ofadministration where access to the target cells by the inhibitor isobtained. Injections can be, e.g., intravenous, intradermal,subcutaneous, intramuscular or intraperitoneal. For example, theinhibitor can be injected intravenously or intramuscularly for treatmentof multiple sclerosis, or can be injected directly into the joints fortreatment of arthritic disease, or can be injected directly into thelesions for treatment of pemphigus vulgaris. In certain embodiments, theinjections can be given at multiple locations. Implantation includesinserting implantable drug delivery systems, e.g., microspheres,hydrogels, polymeric reservoirs, cholesterol matrices, polymericsystems, e.g., matrix erosion and/or diffusion systems and non-polymericsystems, e.g., compressed, fused or partially fused pellets. Inhalationincludes administering the inhibitor with an aerosol in an inhalator,either alone or attached to a carrier that can be absorbed. For systemicadministration, it may be preferred that the inhibitor is encapsulatedin liposomes. Topical administration can be accomplished with, e.g.,ointments, creams or lotions, which are applied topically to theaffected area of the skin. In such compositions, the inhibitor can,e.g., be dissolved in a solvent, and then mixed with, e.g., an emulsionor a gelling agent, as are well known to persons ordinarily skilled inthe art.

In certain embodiments of the invention, the administration can bedesigned so as to result in sequential exposures to the inhibitor oversome time period, e.g., hours, days, weeks, months or years. This can beaccomplished by repeated administrations of the inhibitor by one of themethods described above, or alternatively, by a controlled releasedelivery system in which the inhibitor is delivered to the mammal over aprolonged period without repeated administrations. By a controlledrelease delivery system is meant that total release of the inhibitordoes not occur immediately upon administration, but rather is delayedfor some time period. Release can occur in bursts or it can occurgradually and continuously. Administration of such a system can be,e.g., by long acting oral dosage forms, bolus injections, transdermalpatches and subcutaneous implants.

Examples of systems in which release occurs in bursts include, e.g.,systems in which the inhibitor is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme,and systems in which the inhibitor is encapsulated by anionically-coated microcapsule with a microcapsule core degrading enzyme.Examples of systems in which release of the inhibitor is gradual andcontinuous include, e.g., erosional systems in which the inhibitor iscontained in a form within a matrix, and diffusional systems in whichthe inhibitor permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be, e.g., in the form of pellets orcapsules.

The inhibitor can be suspended in a liquid, e.g., in dissolved form orcolloidal form. The liquid can be a solvent, partial solvent ornonsolvent. In many cases water or an organic liquid can be used.

The inhibitor is administered to the mammal in a therapeuticallyeffective amount. By therapeutically effective amount is meant thatamount which is capable of at least partially preventing, reversing,reducing, decreasing, ameliorating or otherwise suppressing theparticular immune response being treated. A therapeutically effectiveamount can be determined on an individual basis and will be based, atleast in part, on consideration of the species of mammal; the mammal'sage, sex, size, and health; the inhibitor used; the type of deliverysystem used; the time of administration relative to the severity of thedisease; and whether a single, multiple, or controlled release doseregimen is employed. A therapeutically effective amount can bedetermined by one of ordinary skill in the art employing such factorsand using no more than routine experimentation.

Preferably, the concentration of the inhibitor if administeredsystematically is at a dose of about 1.0 mg to about 2000 mg for anadult of 70 kg body weight, per day. More preferably, the dose is about10 mg to about 1000 mg/70 kg/day. Most preferably, the dose is about 100mg to about 500 mg/70 kg/day. Preferably, the concentration of theinhibitor if applied topically is about 0.1 mg to 500 mg/gm of ointment,more preferably is about 1.0 mg to about 100 mg/gm ointment, and mostpreferably is about 30 mg to about 70 mg/gm ointment. The specificconcentration partially depends upon the particular inhibitor used, assome are more effective than others. The dosage concentration of theinhibitor that is actually administered is dependent at least in partupon the particular immune response being treated, the finalconcentration of inhibitor that is desired at the site of action, themethod of administration, the efficacy of the particular inhibitor, thelongevity of the particular inhibitor, and the timing of administrationrelative to the severity of the disease. Preferably, the dosage form issuch that it does not substantially deleteriously affect the mammal. Thedosage can be determined by one of ordinary skill in the art employingsuch factors and using no more than routine experimentation.

III. Inhibitors of Cathepsin S

The present invention employs inhibitors of cathepsin S in a variety ofmethods, as described above, and also provides for a new class of novelpeptide-based cathepsin S inhibitors. Therefore, the present inventionprovides for the use of an inhibitor of cathepsin S in a medicament, orin a pharmaceutical or therapeutic preparation, for inhibiting invariantchain proteolysis from class II MHC/invariant chain complexes, forreducing the competency of class II MHC molecules for binding antigenicpeptides, for reducing presentation of antigenic peptide by class II MHCmolecules, for suppressing a class II MHC-restricted immune response, orfor treating an autoimmune disease, allergic response, or allogeneicimmune response.

The inhibitors of cathepsin S, as used in the methods of the presentinvention, may be regarded as being prior art inhibitors of cathepsin S,the novel peptide-based inhibitors of cathepsin S disclosed herein, orcurrently unknown or undisclosed inhibitors of cathepsin S which, forpurposes of the present invention, are equivalents to the prior art andpresently disclosed inhibitors. Alternatively, and as discussed below,inhibitors of cathepsin S may be regarded broadly as beingnon-peptide-based inhibitors or peptide-based inhibitors, as definedabove.

A Non-Peptide-Based Inhibitors

Non-peptide-based inhibitors of cathepsin S include repressors,antisense sequences, and non-peptide-based competitive andnon-competitive inhibitors.

At present, there are no known repressors of cathepsin S induction ortranscription which satisfy the definition of an inhibitor of cathepsinS as used herein. Nonetheless, upon the discovery of such repressors,their use in the methods and products of the present invention may behighly preferred over currently known inhibitors, and would be regardedas an equivalent embodiment of the disclosed methods and products.

Antisense sequences to cathepsin S may readily be chosen and produced byone of ordinary skill in the art on the basis of the known nucleic acidsequence of the cathepsin S gene (see, e.g., GenBank Accession Nos.M86553, M90696, S39127; and Wiedersranders et al., J Biol. Chem. 267:13708-13713 (1992)) and the developing field of antisense technology Inorder to be sufficiently selective and potent for cathepsin Sinhibition, such cathepsin S-antisense oligonucleotides should compriseat least 10 bases and, more preferably, at least 15 bases. Mostpreferably, the antisense oligonucleotides comprise 18-20 bases.Although oligonucleotides may be chosen which are antisense to anyregion of the cathepsin S gene or mRNA transcript, in preferredembodiments the antisense oligonucleotides correspond to the N-terminalor translation initiation region of the cathepsin S mRNA, or to mRNAsplicing sites. In addition, cathepsin S antisense may, preferably, betargeted to sites in which mRNA secondary structure is not expected(see, e.g., Sainio et al. (1994) Cell. Mol. Neurobiol. 14(5):439-457)and at which proteins are not expected to bind.

As will be obvious to one of ordinary skill in the art, the cathepsinS-inhibiting antisense oligonucleotides of the present invention neednot be perfectly complementary to the cathepsin S gene or mRNAtranscript in order to be effective. Rather, some degree of mismatcheswill be acceptable if the antisense oligonucleotide is of sufficientlength. In all cases, however, the oligonucleotides should havesufficient length and complementarity so as to selectively hybridize toa cathepsin S transcript under physiological conditions. Preferably, ofcourse, mismatches are absent or minimal. In addition, although it isnot recommended, the cathepsin S-antisense oligonucleotides may have oneor more non-complementary sequences of bases inserted into an otherwisecomplementary cathepsin S-antisense oligonucleotide sequence. Suchnon-complementary sequences may loop out of a duplex formed by acathepsin S transcript and the bases flanking the non-complementaryregion. Therefore, the entire oligonucleotide may retain an inhibitoryeffect despite an apparently low percentage of complementarity.

The cathepsin S-antisense oligonucleotides of the invention may becomposed of deoxyribonucleotides, ribonucleotides, or any combinationthereof. The 5′ end of one nucleotide and the 3′ end of anothernucleotide may be covalently linked, as in natural systems, via aphosphodiester internucleotide linkage. These oligonucleotides may beprepared by art recognized methods such as phosphoramidate,H-phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g.,Uhlmann et al. (1990) Chem. Rev. 90:543-584; Agrawal (ed.) Meth. Mol.Biol., Humana Press, Totowa, N.J. (1993) Vol. 20; and U.S. Pat. No.5,149,798) which may be carried out manually or by an automatedsynthesizer (reviewed in Agrawal et al. (1992) Trends Biotechnol.10:152-158).

The cathepsin S-antisense oligonucleotides of the invention also mayinclude modified oligonucleotides. That is, the oligonucleotides may bemodified in a number of ways which do not compromise their ability tohybridize to nucleotide sequences contained within the transcriptioninitiation region or coding region of the cathepsin S gene. The termmodified oligonucleotide as used herein describes an oligonucleotide inwhich at least two of its nucleotides are covalently linked via asynthetic linkage, i.e., a linkage other than a phosphodiester linkagebetween the 5′ end of one nucleotide and the 3′ end of anothernucleotide. The most preferred synthetic linkages are phosphorothioatelinkages. Additional preferred synthetic linkages includealkylphosphonates, phosphorodithioates, phosphate esters,alkylphosphonothioates, phosphoramidates, carbamates, carbonates,phosphate triesters, acetamidate, and carboxymethyl esters.Oligonucleotides with these linkages or other modifications can beprepared according to known methods (see, e.g., Agrawal and Goodchild(1987) Tetrahedron Lett. 28:3539-3542; Agrawal et al. (1988) Proc. Natl.Acad. Sci. (USA) 85:7079-7083; Uhlmann et al. (1990) Chem. Rev.90:534-583; Agrawal et al. (1992) Trends Biotechnol. 10:152-158;Agrawal.(ed.) Meth. Mol. Biol., Humana Press, Totowa, N.J. (1993) Vol.20).

The term modified oligonucleotide also encompasses oligonucleotides witha modified base and/or sugar. For example, modified oligonucleotidesinclude oligonucleotides having the sugars at the most 3′ and/or most 5′positions attached to chemical groups other than a hydroxyl group at the3′ position and other than a phosphate group at the 5′ position. Othermodified ribonucleotide-containing oligonucleotides may include a2′-O-alkylated ribose group such as a 2′-O-methylated ribose, oroligonucleotides with arabinose instead of ribose. In addition,unoxidized or partially oxidized oligonucleotides having a substitutionin one nonbridging oxygen per nucleotide in the molecule are alsoconsidered to be modified oligonucleotides.

Such modifications may be at some or all of the internucleosidelinkages, at either or both ends of the oligonucleotide, and/or in theinterior of the molecule (reviewed in Agrawal et al. (1992) TrendsBiotechnol. 10:152-158 and Agrawal (ed.) Meth. Mol. Biol., Humana Press,Totowa, N.J. (1993) Vol. 20). Also considered as modifiedoligonucleotides are oligonucleotides having nucleaseresistance-conferring bulky substituents at their 3′ and/or 5′ end(s)and/or various other structural modifications not found in vivo withouthuman intervention. Other modifications include additions to theinternucleoside phosphate linkages, such as cholesteryl or diaminecompounds with varying numbers of carbon residues between the aminogroups and terminal ribose.

Other non-peptide-based inhibitors of cathepsin S include antibodies,including fragments of antibodies such as Fc, which selectively bind toand inhibit the activity of cathepsin S; and ribozymes which interferewith the transcription, processing or translation of cathepsin S mRNA.

B. Peptide-Based Inhibitors: Generally

Peptide-based inhibitors of cathepsin S are, at the molecular level,mimics or analogs of at least a portion of a natural polypeptidesequence which binds to the active site of cathepsin S as a substrate.In their simplest form, peptide-based inhibitors of cathepsin S aresimply peptides which are based upon the sequences adjacent to knowncathepsin S cleavage sites. Such peptide-based inhibitors arecompetitive inhibitors. Preferably, however, peptide-based inhibitorshave modified polypeptide structures (whether synthesized from peptidesor not) which alter their activity, stability, and/or specificity. Theart of combinatorial chemistry has progressed significantly in thedesign of peptide-based inhibitors such that it is now routine toproduce large numbers of inhibitors based on one or a few peptidesequences or sequence motifs (see, e.g., Brömme et al., Biochem. J.315:85-89 (1996), Palmer et al., J. Med. Chem. 38:3193-3196. (1995)).Thus, for example, if cathepsin S is known to cleave protein X atposition Y, a peptide-based inhibitor of cathepsin S may be chosen ordesigned as a polypeptide or modified polypeptide having the samesequence as protein X, or structural similarity to the sequence ofprotein X, in the region adjacent to position Y. In fact, the regionadjacent to the cleavage site Y, spanning residues removed by 10residues or, more preferably, five residues N-terminal and C-terminal ofposition Y, may be defined as a “preferred protein X cleavage site” forthe choice or design of peptide-based inhibitors. Thus, a plurality ofpeptide-based inhibitors, chosen or designed to span the preferredprotein X cleavage site around position Y, may be produced, tested forinhibitory activity, and sequentially modified to optimize or alteractivity, stability, and/or specificity.

Preferably, the peptide portion of the peptide-based inhibitors of theinvention can be any length, as long the compound can inhibitproteolysis by cathepsin S. Preferably, the peptide portion is about 2to about 20 amino acids or amino acid equivalents long, more preferablyit is about 2 to about 10 amino acids or amino acid equivalents long,and most preferably it is about 2 to about 3 amino acids or amino acidequivalents long.

Modified peptide-based inhibitors of cysteine proteases, as well asother enzymes, are well known in the art. Thus, for example,peptide-based inhibitors of cysteine proteases include peptidylaldehydes, nitrites, α-ketocarbonyls, halomethyl ketones, diazomethylketones, (acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfoniumsalts, epoxides, and N-peptidyl-O-acylhydroxylamines (see, e.g., Brömmeet al., Biochem. J. 315:85-89 (1996); Palmer et al., J. Med. Chem.38(17):3193-3196 (1995); Brömme et al., Biol. Chem. 375:343-347 (1994);and references cited therein.

Currently preferred peptide-based inhibitors of cathepsin S includethose based upon the sequences Leu-Leu-Leu, and Leu-Hph (where Hphindicates homophenylalanine), as well as the preferred invariant chaincleavage site sequences described below. Thus, for example,morpholinurea-Leu-Hph-vinylsulfone phenyl (LHVS) is one preferredcathepsin S inhibitor. Also preferred are other peptidyl vinyl sulfones,with or without the addition of an N-terminal groups such ascarboxybenzyl or nitrophenylacetyl groups, such as Leu-Leu-Leu-vinylsulfone, N-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone, andN-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone. Most preferably, thepeptidyl moiety corresponds to 2-3 residues chosen from the cathepsin Spreferred invariant chain cleavage site, as described below.

C. Peptide-Based Inhibitors: Novel Cathepsin S Inhibitors

As noted above, and evidenced in the Examples below, cathepsin S cleavesthe invariant chain, while associated in a class II MHC/invariant chaincomplex, at two major, nearly adjacent, locations. These major cleavagesoccur C-terminal of the Arg residue at position 78 or the Lys residue atposition 80 of the human invariant chain sequence (SEQ ID NO: 1). SeeExample 8. Therefore, a preferred invariant chain cleavage site extendsaround Arg₇₈ from N-terminally about positions 68-73 to C-terminallyfrom about positions 83-88. Similarly, a preferred invariant chaincleavage site extends around Lys₈₀ from N-terminally about positions70-75 to C-terminally from about positions 85-90. Because of the overlapof these regions, cathepsin S has a preferred invariant chain cleavagesite spanning, approximately, from N-terminally about positions 68-75 toC-terminally about positions 83-90. Thus, peptide-based inhibitors ofcathepsin S based upon a sequence of 2-20, more preferably 2-10, andmost preferably 2-3 consecutive residues from within this site areprovided.

Because of the postulated nature of the cathepsin S active site (see,e.g., Brömme et al., Biochem. J. 315:85-89 (1996)), it is particularlypreferred that the residues one-, two- and, optionally, three-positionsN-terminal to the cleavage sites be included in a peptide basedinhibitor. Thus, for example, the residues Asn-Leu (positions 76 and 77;or −2 and −1 relative to the Arg₇₈ cleavage point) or the residuesGlu-Asn-Leu (positions 75, 76, and 78; or −3, −2 and −1 relative to theArg₇₈ cleavage point) are preferably used as a basis for choosing ordesigning a peptide-based inhibitor. Similarly, the residues Arg-Met(positions 78 and 79; or −2 and −1 relative to the Lys₈₀ cleavage point)or the residues Leu-Arg-Met (positions 77, 78, and 79; or −3, −2 and −1relative to the Lys₈₀ cleavage point) are preferably used as a basis forchoosing or designing a peptide-based inhibitor.

Thus, for example, the invention provides novel peptide-based inhibitorssuch as vinylsulfones compounds including Asn-Leu-vinylsulfone,Arg-Met-vinylsulfone, Leu-Arg-Met-vinylsulfone,Glu-Asn-Leu-vinylsulfone, and Leu-Leu-Leu-vinylsulfone. Modifications ofthese peptide vinylsulfones are also included in the invention. Forexample, carboxybenzyl can be present at the N-terminal end to give thefollowing compounds: N-(carboxybenzyl)-Asn-Leu-vinylsulfone,N-(carboxybenzyl)-Arg-Met-vinylsulfone,N-(carboxybenzyl)-Leu-Arg-Met-vinylsulfone,N-(carboxybenzyl)-Glu-Asn-Leu-vinylsulfone, andN-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone. In an alternative,nitrophenylacetyl is present at the N-terminal end to give the followingcompounds: N-(nitrophenylacetyl)-Asn-Leu-vinylsulfone,N-(nitrophenylacetyl)-Arg-Met-vinylsulfone,N-(nitrophenylacetyl)-Leu-Arg-Met-vinylsulfone,N-(nitrophenylacetyl)-Glu-Asn-Leu-vinylsulfone, andN-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone. A peptide-basedvinylsulfone is meant to include, e.g., a peptide vinylsulfone or amodified peptide vinylsulfone. The invention is also meant to includeother modifications of the peptide-based vinylsulfones, e.g.,substitutions can be added at the amino terminus of the peptide-basedvinylsulfones by, e.g., N-methyl substituents or any other alkyl orsubstituted alkyl chain, or by substitution with, e.g., phenyl, benzyl,aryl, or modified aryl substituents, as would be known to those skilledin the art.

These examples are merely illustrative and not exhaustive. For example,the peptide-based inhibitors of cathepsin S can be based upon otherpeptide sequences which span a portion of the preferred invariant chaincleavage site, for example, any 2-3, 3-5, 5-7, or more consecutiveresidues within the preferred invariant chain cleavage site. Similarly,the peptide-based inhibitors may be peptidyl aldehydes, nitriles,α-ketocarbonyls, halomethyl ketones, diazomethyl ketones,(acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts,epoxides, N-peptidyl-O-acylhydroxylamines, or other such compounds knownto those of skill in the art. Furthermore, the N-termini of thesepeptide-based inhibitors of cathepsin S may be blocked with a variety ofsubstituent groups, including N-methyl substituents or other alkyl orsubstituted alkyl chains; phenyl, benzyl, aryl, or modified arylsubstituents; or other such substituents known to those of skill in theart.

EXAMPLES Example 1

Active Site Labeling of Cysteine Proteases

This example illustrates the active site labeling of cysteine proteases,including cathepsin S. The role of cathepsin S in the proteolyticprocessing of Ii was investigated by exploiting the properties of anumber of protease inhibitors, both novel and previously described. Thecysteine protease inhibitor Cbz-Tyr-Ala-CN₂, irreversibly binds to theactive site of cysteine proteases in proportion to their activity. Aprofile of the active cysteine proteases present within a given celltype can be directly established by incubating the cells with aniodinated form of this inhibitor, Cbz-[¹²⁵I]-Tyr-Ala-CN₂ and visualizingthe labeled proteases on SDS-PAGE (Mason et al., Biochem. J. 257:125-129(1989)). Cysteine proteases in the cell that are first inactivated withother cysteine class inhibitors prior to incubation withCbz-[¹²⁵I]-Tyr-Ala-CN₂ produce a corresponding decrease in labeling.Inhibition specific for a given protease affects subsequent labelingwith Cbz-[¹²⁵I]-Tyr-Ala-CN₂ of that specific protease; but not otherenzymes present in the preparation.

To examine the cysteine protease profile of professional antigenpresenting cells, and measure specifically the activity of cathepsin S,the B-lymphoblastoid cell line HOM2 (Benaroch et al., EMBO J. 14:37-49(1995)) was used. The B-lymphoblastoid cell line HOM2 /(homozygous forHLA-DR1) was maintained in RPMI with 10% FBS, 1/1000 units/mlpenicillin, 100 /μg/ml streptomycin and 2 mM glutamine. HOM2 cells werelabeled with Cbz-[¹²⁵I]-Tyr-Ala-CN₂ after incubation with 0.1 nM and 5nM of the specific cathepsin S inhibitor, LHVS(morpholinurea-leucine-homophenylaline-vinylsulfone phenyl) (Palmer etal., J. Med. Chem. 38(17):3193-3196 (1995)). Lysates prepared from thelabeled cells were analyzed either directly or subjected toimmunoprecipitation with antibodies specific for cathepsins S and B by120% SDS-PAGE. Antibody to human cathepsin S was prepared as describedin Shi et al., J. Biol. Chem. 269:11530-11536 (1994), and antibody tocathepsin B was obtained from Vital Products, Inc. (St. Louis, Mo.). Inthe absence of cathepsin S inhibitor, three polypeptides were labeled,migrating at 33 kDa, 28 kDa, and 6 kDa (running with the dye front inthis 12% gel). The 33 kDa and 6 kDa polypeptides wereimmunoprecipitation with an antiserum specific for cathepsin B, andrepresent the single-and light-chain forms of the active enzyme,respectively. The more intense labeling of the 6 kDa polypeptidecompared with the 33 kDa protein suggested that most of the cathepsin Bpresent in HOM2 cells was in the light chain form. The 28 kDapolypeptide was identified as the active form of cathepsin S, as it wasimmunoprecipitated with an antibody specific for this enzyme. Thelabeling of cathepsin S, but not cathepsin B, was effectively inhibitedat an LHVS concentration of 1 nM. At an inhibitor concentration of 5 nM,the labeling of cathepsin B was decreased, although some activityremained. Thus, LHVS was able to be utilized at 1-5 nM concentrations tospecifically inhibit cathepsin S in HOM2 cells, leaving other cysteineproteases functionally active.

The active site labeling of cysteine proteases withCbz-[¹²⁵I]-Tyr-Ala-CN₂ was then used to determine that the purifiedhuman cathepsins B and S, used in subsequent experiments were notcross-contaminated with other proteases.

The cysteine protease inhibitor Cbz-Tyr-Ala-CN₂ was iodinated aspreviously reported (Mason et al., Biochem. J. 257: 125-129 (1989)).HOM2 cells (5×10⁻⁶ cells/sample) were incubated with inhibitors2S,3S-trans-epoxysuccinyl-L-Leu-amido-3-methylbutane ethyl ester (E64D)(20 μm) (obtained from Sigma Chemical Co., St. Louis, Mo.), leupeptin(0.5 mM) (obtained from Sigma Chemical Co., St. Louis, Mo.),concanamycin B (20 nM) (obtained from Ajinomoto Co., Kanagawa, Japan),or LHVS (1 or 5 nM) (obtained from Khepri Pharmaceuticals, Inc., SouthSan Francisco, Calif.) at 37° C. for 1 hour prior to labeling. Cellswere labeled by incubation with Cbz-[¹²⁵I]-Tyr-Ala-CN₂ for 1 hour at 37°C., washed twice with PBS and lysed in the SDS-PAGE sample buffer. Thepurified cathepsins B and S were labeled by addition of a 2 μl aliquotof purified enzyme stock to 50 μl of digestion buffer (50 mM Na Acetate,pH 5.5, 1% Triton-X-100, 3 mM cysteine, 1 mM EDTA) containingCbz-[²⁵I-Tyr-Ala-CN₂. Samples were incubated for 1 hour at 37° C. andthe labeling reaction was stopped by the addition of 50 μl of 2×SDS-PAGE sample buffer.

Immunoprecipitation of cathepsins B and S was performed by labeling5×10⁶ HOM2 cells as above followed by cell lysis with 1 ml of 10 mMTris-HCl, pH 7.5, 1 mM EDTA, 0.2. SDS, 1% Triton X-100 on ice. Lysateswere collected, boiled for 5 minutes and precleared with proteinA-agarose (Boehringer Mannheim, Indianapolis, Ind.) plus normal rabbitserum (Sigma Chemical Co, St. Louis, Mo.). The cysteine proteases wereimmunoprecipitated with anticathepsin S antibody and anti-cathepsin Bantibody coupled to protein agarose. The pellets were washed and elutedwith reducing SDS-PAGE sample buffer.

Example 2

Inhibition of Cathepsin S Prevents Ii Processing

This example illustrates that inhibition of cathepsin S interferes withprocessing of Ii and subsequent peptide binding by class II molecules.HOM2 cells were pulsed-labeled with ³⁵S-methionine/cysteine as follows.HOM2 cells, 5×10⁶/sample, were preincubated in 1 ml methionine-,cysteine-free DMEM supplemented with protease inhibitors or appropriatesolvent for 1 hour at 37 C prior to labeling with 0.25 mCi³⁵S-methionine/cysteine for 1 hour at 37 C. The cells were centrifugedand resuspended at 1×10⁶/ml in RPMI/10% FBS and chased for 5 hours inthe absence or presence of 1 nM or 5 nM LHVS. The HOM2 cells were thenwashed twice with cold PBS and lysed in 1 ml of 50 mM Tris-HCl, pH 7.4,0.5% NP-40, 5 MM MgCl₂. Lysates were precleared with protein A-agarose.7 μl of normal rabbit serum and 2 μl of normal mouse serum followed byimmunoprecipitation of class II αβ dimers and αβIi complexes with themonoclonal antibody Tu36 coupled to protein A-agarose.Immunoprecipitates were washed 4-6× with 1 ml of 50 mM Tris-HCl, pH 7.4,0.5% NP-40, 5 mM EDTA, 150 mM NaCl. These pellets were either eluteddirectly with non-reducing or reducing SDS-PAGE sample buffer, or usedas starting material for proteolytic digestion and furtherimmunoprecipitation.

Proteolytic digestion of immunoprecipitates was performed by incubationof precipitated pellets with purified proteases diluted in 50 μl of 50mM Na Acetate, pH 5.5, 1% Triton X-100, 3 mM cysteine, 1 mM EDTA at 37°C. Samples were eluted with SDS-PAGE sample buffer.

One half of the samples were analyzed by 14% SDS-PAGE under mildlydenaturing (non-boiled, nonreduced) conditions to visualize SDS-stablecomplexes which migrate at approximately 50 kDa. These SDS-stablecomplexes represent peptide-loaded αβ-dimers, which decrease uponinhibition of cysteine proteases with leupeptin (Neefjes and Ploegh,EMBO J. 11:411-416 (1992)) (obtained from Sigma Chemical Co., St. Louis,Mo.). Specific inhibition of cathepsin S with 1 nM and 5 nM LHVSresulted in accumulation of a class II-associated 13 kDa Ii fragment,and a concomitant reduction in peptide loading as evidenced by a markeddecrease in formation of SDS-stable complexes. Inhibition of allcysteine proteases with the cysteine-class inhibitor 2S,3S-trans-epoxysuccinyl-L-Leu-amido-3-methylbutane ethyl ester (E64D)resulted in a buildup of a class II-associated 23 kDa Ii fragment with adecrease in SDS-stable dimer formation, similar to that seen withleupeptin. This result indicated that cathepsin S acted on a relativelylate Ii breakdown intermediate and was required for efficientproteolysis of Ii necessary for subsequent peptide loading.

Example 3

Cathepsin S Selectively Digests Ii Participating in αβIi Complexes

This example illustrates that cathepsin S proteolytically digests theinvariant chain in class II MHC/invariant chain complexes, leaving theclass II MHC molecule intact and capable of subsequently bindingantigenic peptide. Individual α, β, and Ii polypeptides were translatedin vitro both separately and together under conditions compatible withcomplex formation (Bijlmakers et al., J. Exp. Med. 180:623-629 (1994)).In vitro translation of α, β and Ii was accomplished as follows: cDNA'sof HLA-DRI α (Larhammar et al., Cell 30:153-161 (1982)) and β chains(Bell et al., Proc. Natl. Acad. Sci. (USA) 82:3405-3409 (1985)), and thecDNA encoding the human p33 Ii (Claesson et al., Proc. Natl. Acad. Sci.(USA) 80:7395-7399 (1983)) were all cloned in pSP72 (Promega, Madison,Wis.) as described previously (Bijlmakers et al., J. Exp. Med.180:623-629 (1994)). The cDNA's were transcribed in vitro eithertogether or separately, using T7 RNA polymerase. RNA was stored in 70%ethanol at −80° C. The optimal amount of RNA utilized was determinedempirically for each separate batch of RNA. The resulting RNA's weretranslated in vitro in rabbit reticulocyte lysate (Flexi, Promega,Madison, Wis.), supplemented with canine microsomes. Translations wereperformed for 90 minutes at 30° C. as previously described (Bijlmakerset al., J. Exp. Med. 180:623-629 (1994)). Upon completion of translationthe microsomes were pelleted by centrifugation for 4 minutes at 12,000rpm, and resuspended in 20 μl of lysis/digestion buffer (50 mM NaAcetate, pH 5.5, 1% Triton X-100, 3 mM cysteine, 1 mM EDTA) with orwithout cathepsin S. Cathepsin S concentrations were 0.19 or 0.38 μM.Purified human cathepsin S was obtained by expression in Sf9 cells usingthe Baculovirus expression system as described (Brömme and McGrath,Protein Sci., in press, 1996). Proteolytic digestion was performed byincubation of the above solubilized microsomes for 4 hours at 37° C.Digestion was stopped by the addition of reducing SDS sample buffer,samples boiled, and analyzed directly by 15% SDS-PAGE under denaturingconditions.

Cathepsin S readily digested the α, βand Ii chains when translatedalone. However, when the α and β chains were translated together so thatdimer formation occurred, they displayed resistance to proteolysis. Whenall three moieties were translated together and digested with cathepsinS, only Ii was degraded, illustrating the marked sensitivity of Ii tocathepsin S digestion. The formation of αβ dimers and αβIi trimers wereconfirmed following each step by immunoprecipitation. Thus, cathepsin Sselectively degrades Ii molecules that are part of αβIi complexes,leaving the αβ dimers intact.

To directly compare the ability of cathepsins S and B to degrade Iiparticipating in αβIi trimers, the activity of the two proteases must bemeasured, independent of their differences in substrate specificity.Normalization to total protein content is misleading because only aportion of the total protein may be active. To overcome this difficulty,the molar activity of the purified cathepsin B (obtained fromCalbiochem-Novabiochem Corp., San Diego, Calif.) and cathepsin Spreparations were measured by using E64D (obtained from Sigma ChemicalCo., St. Louis, Mo.) as an active site titrant with the substrateZ-Phe-Arg-AMC, as previously described (Barrett and Kirschke, Methods inEnzymology, Vol. 80, Proteolytic Enzymes, L. Lorand (ed.), New York,N.Y.: Academic Press, Inc., pp 535-560 (1981)). The activities of theenzymes measured in this manner are independent of the substrate usedbecause E64D irreversibly inhibits both cathepsins S and B on anequimolar basis.

Using the concentrations determined by the above method, the ability ofcathepsins S and B to digest Ii from αβIi heterotrimersimmunoprecipitated from HOM2 cells was determined. HOM2 cells wereincubated in the absence of inhibitor, or in the presence of inhibitorsE64D or LHVS, during a ³⁵ S-methionine/cysteine pulse/chase. Cathepsin Sspecifically degraded intact Ii as well as the 23 kDa and 13 kDa Iiintermediates, while sparing the αβ dimer and the αβ-peptide complexes.In contrast, cathepsin B showed little proteolytic activity againstimmunoprecipitated αβIi trimers or αβIi fragment complexes, even at 100×the molar concentration of cathepsin S. The inability of cathepsin B todigest Ii was not merely a result of the enzyme not being active at theconcentrations used, as evidenced by a slight change in migration of theclass II β chain, suggesting activity of cathepsin B on the cytoplasmicportion of the β chain (Roche and Cresswell, Proc. Natl. Acad. Sci.(USA) 88:3150-3154 (1991)).

A defined intermediate in the maturation of class II molecules is acomplex consisting of the αβ heterodimer bound to the CLIP region of Ii(Avva and Cresswell, Immunity 1:763-774 (1994)). It is this intermediatethat was proposed to be a substrate for HLA-DM (see Wolf and Ploegh,Ann. Rev. Cell Biol. 11:267-306 (1995)). To determine if cathepsin Scould generate αβ-CLIP from αβIi, HOM2 cells were labeled in the absenceand presence of inhibitors, immunoprecipitated with the monoclonalantibody Tu36, digested with 0.23 μM cathepsin S for 1 hour at 37° C.,and immunoprecipitated with antibodies directed against Ii. Tu36 is amouse monoclonal antibody (Shaw et al., Human Immunol. 12:191-211(1985)) which recognizes HLA-DRI αβ dimers alone or in association withIi. Two Ii reactive mouse monoclonal antibodies were used: PIN-1,specific for the N-terminus of Ii (Avva and Cressell, Immunity 1:763-774(1994)), and the monoclonal antibody LN-2, directed against theC-terminus of Ii. Immunoprecipitates were then boiled for 3 minutes inthe presence of 1% SDS to unfold the αβIi complexes, diluted 10-fold andre-immunoprecipitated sequentially with antibodies against the CLIPregion, cytoplasmic tail (PIN-1 antibody), and lumenal domain (LN-2antibody) of Ii. Samples were analyzed by 10-20%. gradient tricineSDS-PAGE under denaturing conditions. A 3 kDa polypeptide wasimmunoprecipitated with the anti-CLIP antibody, which was not found inthe undigested samples. The anti-CLIP reagent was generated by injectionof rabbits with two overlapping peptides, conjugated to KLH, spanningthe region of residues 81-104 of intact human Ii. The antibody directedagainst the N-terminal cytoplasmic tail, PIN-1, was able toimmunoprecipitate intact Ii as well as the 23 kDa and 13 kDa Ii chainfragments, suggesting that both the 23 kDa and 13 kDa intermediates areN-terminal fragments. The LN2 antibody against the Ii lumenal domainprecipitated only the full length Ii. Thus, cathepsin S was able toproduce αβ-CLIP, a known intermediate in αβIi proteolysis.

In contrast to cathepsin S, neither purified cathepsin B (66 μM) nor D(2.4 μM) (obtained from Calbiochem-Novabiochem Corp., San Diego, Calif.)could produce αβ-CLIP from αβIi. HOM2 cells were pulse/chased in thepresence of 0.5 mM leupeptin and class II molecules wereimmunoprecipitated with Tu36. The isolated class II complexes were thendigested with the different purified cathepsins at pH 5.5 for 1 hour at37° C. The digested complexes were then analyzed by 10-20%; tricine gelunder denaturing conditions. Digestion with cathepsin S (0.23 μM) aloneresulted in the production of a 3 kDa polypeptide associated with αβdimers. This 3 kDa fragment could also be re-immunoprecipitated with theanti-CLIP reagent used above. Both cathepsins B and D, when used at highconcentrations, could produce large molecular weight Ii cleavageproducts, but not up-CLIP, illustrating the essential role of cathepsinS in complete and efficient Ii processing.

While inclusion of HLA-DM facilitates exchange of CLIP bound to theαβ-CLIP complex for antigenic peptide (Denzin et al., Cell 82:155-165(1995); Sherman et al, Immunity 3:197-205 (1995); Sloan et al., Nature375:802-806 (1995)), the reaction can also occur, albeit lessefficiently, in the absence of added HLA-DM. The displacement of largerIi remnants with antigenic peptides is even less efficient. Proteolysisof αβIi heterotrimers with cathepsin S was shown to allow peptideloading onto the resulting class II αβ-CLIP complexes as follows: HOM2cells were treated with 20 nM concanamycin B to accumulate a largeamount of αβIi trimers (Benaroch et al., EMBO J. 14(1):37-49 (1995)).The hemagglutinin peptide (HA), containing amino acids 306-318, was usedas a DR1 restricted peptide (Rothbard et al., Cell 52(4):515-523(1988)). HA was synthesized (t-boc chemistry) on a Biosearch SAM 2peptide synthesizer, dissolved in water and stored at −70° C. The αβIicomplexes from concanamycin B treated cells were initiallyimmunoprecipitated with an antibody against the lumenal domain (LN-2) toprecipitate only intact αβIi complexes. These complexes were incubatedin the absence and presence of 0.23 μM cathepsin S at pH 5.5, and thenexposed to ¹²⁵I-HA at pH 5.5 for 4 hours. After removal of unbound¹²⁵I-HA, complex formation was assessed by 14% SDS-PAGE under mildlydenaturing (nonboiled, nonreduced) conditions. HA peptide was iodinatedby incubation of HA (50 μl of 1 mM solution) with ¹²⁵I and 50 mM NaPO₄(20 μl), pH 7.5 in an iodogen-coated glass tube on ice for 10 minutes.¹²⁵I-HA was separated from free ¹²⁵I by passage over a C18 Sep-packcolumn, and eluted with acetonitrile. Aliquots of ¹²⁵I-HA were dried ina speed-vac and redissolved in digestion buffer for incubation withcathepsin S treated and nontreated immunoprecipitates. Followingincubation with the peptide, samples were diluted to 0.8 ml with lysisbuffer, pH 7.4, cleared of LN-2 antibody with protein A-agarose andimmunoprecipitated with Tu36. Samples were washed thoroughly prior toaddition of SDS-PAGE sample buffer to remove unbound peptide.

Digestion of αβIi with cathepsin S and subsequent exposure to ¹²⁵I-HAresulted in the formation of labeled αβ-peptide complexes, although thisconversion was incomplete as evidenced by the continued presence ofSDS-labile class II molecules. Cathepsin S is thus able to process Iiwhile leaving the class II molecules functionally intact, and shows thatcathepsin S is sufficient, by itself, to effectively degrade Ii in amanner that renders αβ-dimers capable of binding peptide.

Example 4

Inhibition of Cathepsin D Does Not Affect Ii Processing

This example illustrates that neither cathepsin D nor cathepsin H arerequired for Ii processing. Maric et al., Proc. Natl. Acad. Sci: (USA)91:2171-2175 (1993), have implicated the aspartyl protease cathepsin Din an early step of Ii breakdown. This question was examined byutilizing a potent aspartyl-class protease inhibitor, CGP 53437 (Alteriet al., Antimic. Ag. Chemotherap. 37:2087-2092 (1993)) which inhibitscathepsin D in the nanomolar range. CGP 53437 inhibits cathepsin Dactivity in human monocytes by 75% and 90% at concentrations of 5 μM and50 μM, respectively. HOM2 cells were labeled with³⁵S-methionine/cysteine and chased for 5 hours without inhibitor, in thepresence of 0.5 mM leupeptin, 5 nM LHVS, 5 μM CGP 53437 or 50 μM CGP53437. Samples were analyzed by 14% SDS-PAGE under mildly denaturingconditions. Inhibition of cathepsin D (obtained fromCalbiochem-Novabiochem Corp., San Diego, Calif.) with CGP 53437 did notresult in accumulation of Ii fragments nor did it produce a decrease inSDS-stable complexes, suggesting that cathepsin D is not essential forIi processing

Cathepsin H, a lysosomal cysteine protease with good aminopeptidase butweak endopeptidase activity, is upregulated by γ-interferon in mouseperitoneal macrophages (Lafuse et al., J. Leuk. Biol. 57:663-669(1995)). Purified human cathepsin H (obtained fromCalbiochem-Novabiochem Corp., San Diego, Calif.) was not inhibited withLHVS nor did it display any proteolytic activity againstimmunoprecipitated αβIi, implying that cathepsin H is not an essentialprotease for Ii degradation.

Example 5

Synthesis of N-(Carboxybenzyl)-Leu-Leu-Leu Vinylsulfone

This example illustrates the steps in the synthesis ofcarboxybenzyl-Leu-Leu-Leu-vinylsulfone.

a) Weinreb amide of Boc-Leucine

A dichloromethane solution of Boc-Leucine-H₂O (1 equiv.), triethylamine(2 equiv.), and PyBOP (1 equiv.), was stirred for several minutes atroom temperature before HCl-N,O-dimethylhydroxylamine (2 equiv.) wasadded dry, along with additional triethylamine (2 equiv.). Afterstirring overnight, this reaction mix was diluted with dichloromethane,then washed twice each with 3N HCl, saturated NaHCO₃ and saturated NaCl.The dichloromethane phase was dried (over MgSO₄) and roto-evaporated,then the product was purified by silica gel flash column chromatography(hexane/ethylacetate, 3:1):

b) Boc-Leucinal

The Weinreb amide of BocLeucine was dissolved in dry Et₂O and added bycannula to an Et₂O suspension of an equimolar amount of LiAlH₄ at 0° C.After stirring for one hour, the reaction flask was removed from 0° C.and allowed to warm to room temperature. After a reaction time of 80minutes, an aqueous solution of KHSO₄, (2 equiv. of KHSO₄, 5 mlH₂O/mmnol of Weinreb amide) was added slowly while stirring. The aqueousphase was extracted with Et₂O and the confined Et₂O phases were washedwith 3N HCl, saturated NaHCO₃, and saturated NaCl. After drying (MgSO₄,)and rotary evaporation, the oil was found to contain one compound bythin layer chromatography.

c) Phosphonate Sulfone

To a stirring room temperature dioxane solution of diethylmethylthiomethyl phosphonate (1 equiv.) was added 5 equiv. of peraceticacid. After stirring for over 3 hours, Pt on carbon was added to quenchunreacted peroxides. The reaction was quenched by the addition of water(10 ml/mmol of the phosphonate sulfide) and the product was extractedwith ethylacetate. After drying (MgSO₄) and roto-evaporation, thephosphonate sulfone was purified by recrystallization from ethylacetateusing a small amount of hexane.

d) Boc-Leu-Vinylsulfone

To a stirring room temperature tetrahydrofuran solution of NaH (2equiv.) under Argon, was added 2 equiv. of phosphonate sulfone intetrahydrofuran, by cannula. After stirring for a few minutes, 1 equiv.of Boc-Leucinal in tetrahydrofuran was added by cannula. After 3 hoursthe reaction was quenched with water and the aqueous phase was extractedwith dichloromethane. The combined organic phases were back extractedwith saturated NaCl, dried (MgSO₄), and roto-evaporated to a yellow oil.The product was purified by flash column chromatography(ethylacetate/hexane, 1:1) to yield an oil after lyophilization.

e) Leu-Vinylsulfone Tosylate Salt

An Et₂O solution of anhydrous p-Tosic acid (3 equiv.) was added to dryoil of Boc-Leu-vinylsulfone (1 equiv.) and stirred at room temperatureovernight. The product formed a white precipitate which was recovered bycentrifugation.

f) Carboxybenzyl-Leu-Leucine

To three volumes of a stirring room temperature H₂O/NaHCO₃ solution ofLeucine (1.5 equiv.) was added one volume of dioxane solution ofcarboxybenzyl-Leu-Osu (1 equiv.), and was stirred overnight. The productwas precipitated by addition of aqueous sodium citrate, and then wasextracted by ethylacetate. The combined ethylacetate phases were dried(MgSO₄,) and roto-evaporated to a clear oil. The product was purified byflash column chromatography (dichloromethane/MeOH, 12:1, with 1% TFA).

g) Carboxybenzyl-Leu-Leu-Leu-Vinylsulfone

To a stirred tetrahydrofuran solution of carboxybenzyl-Leu-Leucine (1equiv.), at −10° C., under Argon, was added 1 equiv. each ofN-methylmorpholine and isobutyl chloroformate. After a few minutes, atetrahydrofuran suspension of 1 equiv. each of Leu-vinylsulfone tosylateand N-methylmorpholine, was added and stirring at −10° C. was continuedfor 75 minutes. The reaction was quenched by the addition of 3N HCl andthe product was extracted with dichloromethane. After flash columnchromatography (ethylacetate/hexane, 3:2) the product was recrystallizedfrom warm ethanol.

Example 6

Synthesis of Other Peptide-Based Vinylsulfones

This example illustrates the steps in the synthesis of peptidevinylsulfones and modified peptide vinylsulfones.

(i) Asn-Leu-vinylsulfone

This compound is synthesized as described in Example 5 except that instep (g), asparagine is used instead of carboxybenzyl-Leu-leucine.

(ii) Arg-Met-vinylsulfone

This compound is synthesized as described in Example 5 except that instep (g), arginine is used instead of carboxybenzyl-Leu-leucine, andthat in step (a) Boc-L-methionine is used and converted to Boc-methionalas in (b) and used in steps (d) and (e).

(iii) Leu-Arg-Met-vinylsulfone

This compound is synthesized as described above for compound (ii),except that Leu arginine is used in step (g).

(iv) Glu-Asn-Leu-vinylsulfone

This compound is synthesized as described above for compound (i), exceptthat Glu-asparagine is used in step (g).

(v) N-(carboxybenzyl)-Asn-Leu-vinylsulfone

This compound is synthesized as described above for compound (i), exceptthat carboxybenzyl-protected asparagine is used.

(vi) N-(carboxybenzyl)-Are-Met-vinylsulfone

This compound is synthesized as described above for compound (ii),except that carboxybenzyl-protected arginine is used.

(vii) N-(carboxybenzyl)-Leu-Arg-Met-vinylsulfone

This compound is synthesized as described above for compound (iii),except that carboxybenzyl-protected Leu arginine is used.

(viii) N-(carboxybenzyl)-Glu-Asn-Leu-vinylsulfone

This compound is synthesized as described above for compound (iv),except that carboxybenzyl-protected Glu-asparagine is used.

(ix) N-(nitrophenylacetyl)-Asn-Leu-vinylsulfone

The compound is synthesized as described above for compound (v), exceptthat nitrophenylacetyl-protected asparagine is used.

(x) N-(nitrophenylacetyl)-Arg-Met-vinylsulfone

This compound is synthesized as described above for compound (vi),except that nitrophenylacetyl-protected arginine is used.

(xi) N-(nitrophenylacetyl)-Leu-Arg-Met-vinylsulfone

This compound is synthesized as described above for compound (vii),except that nitrophenylacetyl-protected Leu-arginine is used.

(xii) N-(nitrophenylacetyl)-Glu-Asn-Leu-vinylsulfone

This compound is synthesized as described above for compound (viii),except that nitrophenylacetyl-protected Glu-asparagine is used.

Example 7

Inhibition of Cathepsin S Alters Immune Response

This example illustrates that inhibition of cathepsin S altered theimmune response to tetanus toxoid. Peripheral blood mononuclear cells(PBMC's) were isolated from a tetanus immune individual and cultured inthe presence of tetanus toxoid (250 ng/cc), cysteine-class proteaseinhibitor E64D (20 μM) (obtained from Sigma Chemical Co., St, Louis,Mo.) and specific cathepsin S inhibitor LHVS(morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) (10-20 nM)obtained from Khepri Pharmaceuticals, Inc., South San Francisco, Calif.)for 3-5 days. T cell proliferation was assayed by ³H-thymidine uptakeduring the final 18 hours of culture. In the presence of tetanus toxoid(obtained from Sigma Chemical Co., St. Louis, Mo.), ³H-thymidine uptakeincreased more than four-fold. Both E64D and LHVS attenuated thisresponse by approximately 60%. Thus, specific inhibition of cathepsin Smodulated an immune response. The fact that the attenuation of T cellstimulation was of the same magnitude as that produced by completeinhibition of all cysteine proteases supports the conclusion thatcathepsin S is the major cysteine protease involved in invariant chainprocessing and subsequent antigen presentation.

Example 8

Specific Invariant Chain Cleavage Sites for Cathepsin S

This example illustrates the specific cleavage sites for cathepsin S atthe N-terminus of CLIP. HOM2 cells (Benaroch et al., EMBO J. 14:3749(1995)) were pulse/chased with 5 mCi ³⁵S-methionine for 5 hours in thepresence of 20 mM E64D, lysed, and class II MHC moleculesimmunoprecipitated with mAb Tu36. The immunoprecipitates were digestedwith 0.23 μM cathepsin S for 60 minutes at 37° C., pH 5.5. Class II MHCmolecules were repelleted, analyzed on a 10-20% tricine gel andtransferred to nylon membrane. A radioactive band migrating at 3 kDa wasexcised from the membrane and subjected to radiosequencing. A high levelof counts was found in the first cycle corresponding to the methionineat invariant chain residue 79. A smaller increase in counts was alsoevident at cycle II indicating that a proportion of the peptides presenthad an N-terminus at the leucine residue 81. Thus, bipeptide andtripeptide-vinylsulfones spanning these cleavage sites are excellentcandidates for inhibition of cathepsin S-mediated invariant chainprocessing in vivo.

Example 9

Treating Multiple Sclerosis with Leu-Arg-Met-Vinylsulfone

This example illustrates a method for treating multiple sclerosis in ahuman with a peptide-based vinylsulfone which inhibits proteolysis ofthe invariant chain by cathepsin S. The patient is givenLeu-Arg-Met-vinylsulfone by injection intravenously, once a day. Thedose is 500 mg/70 kg body weight. This treatment alleviates the effectsof multiple sclerosis in the patient.

Example 10

Treating Pemphigus Vulgaris with N-(Carboxybenzyl)-Arg-Met-Vinylsulfone

This example illustrates a method for treating pemphigus vulgaris in ahuman with a peptide-based vinylsulfone which inhibits proteolysis ofthe invariant chain by cathepsin S.N-(carboxybenzyl)-Arg-Met-vinylsulfone is topically applied in the formof an ointment to the lesions resulting from the disease. The dose is 50mg/gm of ointment and is applied daily to the affected area. Thistreatment alleviates the effects of pemphigus vulgaris in the patient.

Those skilled in the art will be able to ascertain, using no more thanroutine experimentation, many equivalents of the specific embodiments ofthe invention described herein. These and all other equivalents areintended to be encompassed by the following claims.

1 217 amino acids amino acid <Unknown> linear protein Protein 1..217/note= “Invariant chain protein” 1 Met Asp Asp Gln Arg Asp Leu Ile SerAsn Asn Glu Gln Leu Pro Met 1 5 10 15 Leu Gly Arg Arg Pro Gly Ala ProGlu Ser Lys Cys Ser Arg Gly Ala 20 25 30 Leu Tyr Thr Gly Phe Ser Ile LeuVal Thr Leu Leu Leu Ala Gly Gln 35 40 45 Ala Thr Thr Ala Tyr Phe Leu TyrGln Gln Gln Gly Arg Leu Asp Lys 50 55 60 Leu Thr Val Thr Ser Gln Asn LeuGln Leu Glu Asn Leu Arg Met Lys 65 70 75 80 Leu Pro Lys Pro Pro Lys ProVal Ser Lys Met Arg Met Ala Thr Pro 85 90 95 Leu Leu Met Gln Ala Leu ProMet Gly Ala Leu Pro Gln Gly Pro Met 100 105 110 Gln Asn Ala Thr Lys TyrGly Asn Met Thr Glu Asp His Val Met His 115 120 125 Leu Leu Gln Asn AlaAsp Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly 130 135 140 Ser Phe Pro GluAsn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile 145 150 155 160 Asp TrpLys Val Phe Glu Ser Trp Met His His Trp Leu Leu Phe Gly 165 170 175 ArgMet Ser Arg His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro 180 185 190Lys Glu Ser Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly Val Thr 195 200205 Lys Gln Asp Leu Gly Pro Val Pro Met 210 215

What is claimed is:
 1. An inhibitor of cathepsin S comprising apeptide-based inhibitor of cathepsin S, wherein said peptide-basedinhibitor of cathepsin S is based upon a peptide sequence whichcomprises at least about 2-20 consecutive residues from a preferredinvariant chain cleavage site of cathepsin S, said preferred invariantchain cleavage site spanning from about residue 68 to about residue 90of the invariant chain amino acid sequence of SEQ ID No.:1.
 2. Aninhibitor of cathepsin S comprising a peptide-based inhibitor ofcathepsin S, wherein said peptide-based inhibitor of cathepsin S isbased upon a peptide sequence which comprises at least about 2-20consecutive residues from a preferred invariant chain cleavage site ofcathepsin S, and wherein said peptide sequence is selected from thegroup consisting of Asn-Leu, Glu-Asn-Leu, Arg-Met, Leu-Arg-Met,Leu-Leu-Leu, and Leu-Hph.
 3. An inhibitor as in claim 1 wherein saidpeptide sequence is selected from the group consisting of Asn-Leu,Glu-Asn-Leu, Arg-Met, Leu-Arg-Met, Leu-Leu-Leu, and Leu-Hph.
 4. Aninhibitor as in claim 1 or 2 wherein said peptide-based inhibitor is apeptide-based vinylsulfone or a modified peptide-based vinylsulfone. 5.An inhibitor as in claim 1 or 2 wherein said peptide-based inhibitor isselected from the group consisting of peptidyl aldehydes, nitrites,α-ketocarbonyls, halomethyl ketones, diazomethyl ketones,(acyloxy)-methyl ketones, vinyl sulfones, ketomethylsulfonium salts,epoxides, and N-peptidyl-O-acyl-hydroxylamines.
 6. An inhibitor as inclaim 1 or 2 wherein said inhibitor is selected from the groupconsisting of Asn-Leu-vinylsulfone, Arg-Met-vinylsulfone,Leu-Arg-Met-vinylsulfone, Glu-Asn-Leu-vinylsulfone, andLeu-Leu-Leu-vinylsulfone.
 7. An inhibitor as in claim 1 or 2 whereinsaid inhibitor is selected from the group consisting ofN-(carboxybenzyl)-Asn-Leu-vinylsulfone,N-(carboxybenzyl)-Arg-Met-vinylsulfone,N-(carboxybenzyl)-Leu-Arg-Met-vinylsulfone,N-(carboxybenzyl)-Glu-Asn-Leu-vinylsulfone, andN-(carboxybenzyl)-Leu-Leu-Leu-vinylsulfone.
 8. An inhibitor as in claim1 or 2 wherein said inhibitor is selected from the group consisting ofN-(nitrophenylacetyl)-Asn-Leu-vinylsulfone,N-(nitrophenylacetyl)-Arg-Met-vinylsulfone,N-(nitrophenylacetyl)-Leu-Arg-Met-vinylsulfone,N-(nitrophenylacetyl)-Glu-Asn-Leu-vinylsulfone, andN-(nitrophenylacetyl)-Leu-Leu-Leu-vinylsulfone.
 9. An inhibitor as inclaim 1 or 2 wherein said inhibitor is formulated as a pharmaceutical ortherapeutic preparation suitable for administration to mammalian cellsin vivo or in vitro.